The development of high-sensitivity, high-selectivity detection formats for chemical and biological molecules is of paramount importance for realizing the full potential of genomics and proteomics advances made over the past decade.1-4 High density gene chips have made it possible to monitor the levels of expression of thousands of genes simultaneously. Lower density chips have shown promise for both laboratory and clinical identification of many potential biohazards in one sample. Although the core accepted and utilized labeling technology is currently based upon molecular fluorophore markers, recent advances in nanoparticle technology have pointed toward systems with significantly higher sensitivities and selectivities and potentially more straightforward and versatile readout hardware than conventional fluorescence-based approaches.5-17 A strong argument is being made for nanoparticles as the next generation labeling technology for biodiagnostic research.
One of the most sensitive and selective detection formats for DNA relies on oligonucleotide-functionalized nanoparticles as probes, a particle-initiated silver developing technique for signal enhancement, and a flatbed scanner for optical readout.8 The current demonstrated detection limit for this “scanometric DNA detection” format is 100 aM, and the utility of the system has been demonstrated with short synthetic strands, PCR products, and genomic DNA targets.17,18 A limitation of this approach is that it is inherently a one color system based upon grey scale. The flexibility and applicability of all DNA detection systems benefit from access to multiple types of labels with addressable and individually discernable labeling information. In the case of fluorescence, others have demonstrated that one can use multiple fluorophores, including quantum dots, to prepare encoded structures with optical signatures that depend upon the types of fluorophores used and their signal ratio within the probes.11,19 These approaches typically use micron size probes so that they can obtain encoded structures with the appropriate signal intensities and uniformities. Moreover, in the case of molecular fluorophores, due to overlapping spectral features and non-uniform fluorophore photobleaching rates,1,11 this approach has several potential complications.
The art describes the use of Surface Enhanced Raman Spectroscopy (SERS) to detect various analytes. For example, U.S. Pat. No. 5,306,403 describes a method and apparatus for DNA sequencing using SERS. U.S. Pat. No. 5,266,498 describes the use of SERS to detect analytes in general. U.S. Pat. No. 5,445,972 describes the use of a Raman label bound to a specific binding molecule. U.S. Pat. No. 5,376,556 describes the use of SERS in immunoassays. U.S. Pat. No. 6,127,120 describes the use of SERS, the detection of nucleic acid and nucleic acid subunits. U.S. Pat. Nos. 6,242,264 and 6,025,202 describe the use of silver to form a SERS active substrate to enhance Raman scattering of adsorbed molecules. None of the previous SERS-based detection methodologies were demonstrated using single or multiplexed sandwich hybridization assay formats. This absence may be due, in part, to the difficulty in reproducibly generating and functionalizating stable SERS-active substrates23 as well as the lack of an appropriate probe design strategy to enable multiplexed detection. Accordingly, there is a need for probes and methods for use in SERS-based detection assays, particularly in single or multiplexed sandwich hybridization assay formats.
The present invention provides a novel agent detection reagent comprising a particle comprising a Raman label and specific binding members bound to the particle for use in SERS-based assays of analytes. In the presence of a target analyte, a substrate containing a capture probe for the analyte, and the detection reagent, the reagent advantageously complexes or binds to the binding partner analyte to form a complex which directly or indirectly binds to the support. The Raman label in the labeled complex on the support can then be SERS activated by staining, for example, silver, gold or copper enhancement to achieve a SERS effect when irradiated with a laser. Generally this complex is captured on a solid support and treated with silver to provide a SERS effect. Alternatively, the complex can be directly or indirectly complexed with an analyte which has already been bound directly or indirectly to a solid support substrate. In the present invention, the SERS effect is produced near the time it is measured. This reagent can advantageously include multiple different Raman dyes bound to the particle carriers as a way of distinguishing particular carriers with particular specific binding members, and as a way of indexing a vast number of reagent for multiplex application.
The invention also provides a detection reagent comprising a conjugate of several different Raman dyes bound to a specific binding substance such as DNA, RNA, polypeptide, antibody, antigen, small molecules, etc. This also serves as a reagent indexing tool.
The invention is particularly distinguished from the prior art method in that the SERS technology is used in conjunction with nanoparticle assay techniques to provide extraordinary sensitivity and specificity of detection of analytes which is particularly amenable to multiplexed determination of analytes.